JPS60108753A - Multilayer chemical analysis element - Google Patents

Abstract

PURPOSE:To enhance accuracy in analysis, by including the fine powder of titanium dioxide, which has not undergone specified surface treatment, in a light shielding layer on a reagent layer, thereby eliminating errors caused by the interfering action of a fluoride. CONSTITUTION:A reagent layer, a light shielding layer, and a porous developing layer are provided in this order on a supporting body having water impermeability and light transmitting property such as a transparent polymer sheet characterized by poor hydrophilic property such as cellulose acetate. The reagent layer is prepared by including a hydrogen peroxide indicator, which yields the change that can be detected under the presence of peroxidase and hydrogen peroxide, peroxidase, and oxidase in a polymer binder. The fine powder of titanium dioxide, which has not undergone surface treatment including aluminum oxide or the hydrated oxide of aluminum, or the fine powder of titanium dioxide, which has not undergone surface treatment, is included in the light shielding layer characterized by oxygen permeability and protein impermeability on the reagent layer. Thus interference by fluoride can be eliminated.

Description

[Detailed description of the invention] [Industrial field of application J] The present invention relates to multilayer chemical analysis,
or in biological fluids, especially blood samples (whole surface, plasma or serum) containing fluoride as a preservative, the interference (or interference) of fluoride with the standard IIi analysis of the analyte has been eliminated. It concerns multilayer chemical analysis elements.

[Prior Art] It is widely practiced to add fluoride, such as sodium fluoride, lithium fluoride, etc., as a preservative to a blood sample (hereinafter referred to as whole surface, plasma, or dead surface unless otherwise specified). It is being said. In addition to its anticoagulant effect on surface fluid11 as a value checking effect, fluoride also has a glycolysis inhibiting effect11, so
It is particularly suitable for addition to a blood sample when measuring grape il+-containing blood in blood. (Original author by Izumi Kanai, edited by Masamitsu Kanai! A “Clinical Examination: Summary” Revised 291 Doctors (Metal Publishing, published in 1983) 228 pages, R, D, Henry,
D, C, Cannon, J, W, Winke1ma
Part nX Clinical Chemistry P
r1nciples and TechnicsJ 2
nd Ed, (Harper & Row, Pub
lishers, published in 1974) 385-388
(See pages, etc.) Fluoride as a preservative is generally added to blood immediately after blood collection, or by collecting blood into a blood sampling tube coated with fluoride. be. Fluoride, for example in the case of NaF, is often contained in a range of about 1 mg to about 10 mg per 1 m blood sample. In methods that use reagent systems containing enzymes, especially oxidases (e.g., glucose oxidase, cholesterol oxidase, etc.), which have become popular in recent years, fluorine anions have an interfering effect that causes negative errors that lower the content of the test substance ( or interference effects). Particularly when performing an analysis using a blood test sample 1 containing fluoride, and when applying a blood test sample 1 containing fluoride to a multilayer analysis element of a dry majestic horn. , 弗7 also has a significant negative error caused by anions, so solving the negative ko'+X is a serious problem.

In order to eliminate the negative error caused by fluoride,
827753, a reagent layer containing a glucose A111 reagent M1 composition containing glucose oxidase, peroxidase, 4-aminoantipyrine, and 7-hydroxy-1-171. In addition, under the conditions of use (analytical conditions 1), 3,3-dimethylglutaric acid, 3,3-dimethylglutaric acid, (It has been proposed to contain chuu or malic acid. Also, in the multilayer analytical element described in Japanese Patent Application No. 131750/1989, negative error X due to fluoride has been proposed.
In order to eliminate the It has been proposed to use calcium acetate or the like containing calcium cations that can form poorly soluble salts. However, in multilayer analytical elements for glucose quantitative analysis to which these technologies are applied, a light shielding layer containing fine titanium dioxide powder whose surface is treated with a substance containing aluminum oxide or a hydrous oxide of aluminum, which has been conventionally used, or When a light reflective layer is provided, not only does a negative error still occur due to fluoride, but in a multilayer analytical element for glucose quantitative analysis that uses calcium acetate etc. It has been found that in a range where a positive error due to fluoride occurs and the amount of fluoride anastomosis 41 is large, a complex interference effect (or interference effect) due to fluoride appears in which a negative error due to fluoride occurs.

[Objective of the Invention] The object of the present invention is to use an oxidase, a peroxidase, a hydrogen donor (chromogen), and a coupler (or a chromogen that develops or changes color by oxidation instead of the M1 combination of the hydrogen donor and the coupler). A multilayer chemical analysis element having one or more reagent layers containing a hydrogen donor compound (a hydrogen donor compound), a light shielding layer (or a light reflection layer) containing fine titanium dioxide powder, and a porous spreading layer. The objective is to eliminate the negative error or 1.7′l difference that occurs as a result of the interfering effect (or 1° interfering effect) due to the fluoride contained in Fl, and to improve the analytical precision by eliminating the It is also possible to improve the accuracy of analysis by maintaining the PH value under the analysis conditions of the sublayer represented by the layer to the intended value (fi).

[Structure of the Invention] The above object is to use aluminum oxide (alumina) or a hydrous oxide of aluminum (aluminum oxide) as a fine titanium oxide powder contained in a light shielding layer (or light reflection layer) in a multilayered powder analytical element. This can be achieved by using fine titanium dioxide powder that has not been surface-treated with a substance containing a chemical compound (e.g., permanent compound) or fine titanium dioxide powder that has not been surface-treated.

The present invention provides a multilayer chemical analysis element in which a reagent layer, a light-shielding layer, and a porous development layer are provided in this order on a water-impermeable, light-transmitting support, in which IMf light-shielding layered aluminum or This is a multilayer chemical analysis element containing fine titanium oxide powder or fine titanium dioxide powder that has not been surface-treated with a substance containing a hydrous oxide of aluminum.

Examples of the water-impermeable, light-transmitting support that can be used in the present invention include Japanese Patent Publication No. 53-21677 and Japanese Patent Application Laid-Open No. 55-1
It can be appropriately selected and used from those used in multilayer 4fr J supports described in specifications such as No. 64356. Examples of supports include less hydrophilic or hydrophobic polymers such as cellulose acetate, cellulose acetate, poly(ethylene terephthalate), polycarbonate of bisphenol A, polystyrene, polymethyl methacrylate, etc. from about 50 JLm. Approximately 1m
m, preferably a transparent film or sheet with a thickness ranging from about 801 Lm to about 400 gm, and a thickness of about 10 gm.
Examples include transparent glass plates having a thickness ranging from 0 gm to about 2 mm, and/or preferably from about 150 gm to about 1 mm. The surface of the support may be subjected to known physicochemical treatment (
For example, ultraviolet rays! Hydrophilic polymers are subjected to (irradiation, corona discharge treatment, glow discharge treatment, etc.) to increase the contact force with the reagent layer, or to physicochemical treatment (or without). A coating layer of gelatin or the like can be provided to increase the contact force with the reagent layer and the like.

The reagent layer comprises a hydrogen peroxide indicator, peroxidase, and oxidase dispersed or dissolved in a polymeric binder having a hydrophilic film-forming surface that produces a detectable change in the presence of peroxidase and hydrogen. This is the layer that is included. As described below, the oxidase layer can be provided as a separate layer and contain oxidase, or both the reagent layer and the oxidase layer can contain oxidase. As a hydrogen peroxide indicator, rAnnales of C11nic
al Chemistry J, 6.24-27(1
989), U.S. 41FA No. 3992158, Special Publication No. 5
5-25840, Japanese Patent Publication No. 56-45599, Japanese Patent Publication No. 58-18628, Japanese Patent Application No. 57-165233, etc., combinations of hydrogen donor monomers (chromogens) and phenol or naphthol couplers, Triaryl imidatool-based leuco dyes described in Japanese Publication No. 57-5519, Japanese Patent Application No. 58-68009, etc., Japanese Patent Publication No. 56-45599,
It is possible to use a dye precursor compound described in Japanese Patent Publication No. 58-18628, etc., which is a single compound and develops or changes color by self-coupling in the presence of peroxidase and hydrogen peroxide. Examples of preferred hydrogen peroxide indicators include the following compounds.

Combination of hydrogen donor (chromogen) and coupler: [Hydrogen donor] 4-aminoantipyrine, 4-amino-2-methyl-3-phenyl-1-(2°4.6-)lichlorophenyl)- 4-amine antipyrine homologues or derivatives such as 3-pyrazolin-5-one [couplers] 1.7-hydroxynaphthalene, ■-
1-hydroxynaphthalene derivatives such as sodium (or potassium) hydroxynaphthalene-2-sulfonate Triarylimidazole leuco dye: 4.5-bis[4-(dimethylamino)phenyl]-2-(4-hydroxy-3, Dye precursor compounds such as 5-dimethoxyphenyl)imidazole, 4-(dimethylamino)phenyl-2-(4-hydroxy-3,5-dimethoxyphenyl)-5-phenethylimidazole; dianisidine, 4-7toxy-1-
As peroxidase such as naphthol, Japanese Patent Publication No. 56-45599,
Peroxidases of plant and animal origin (EC1, 11, 1, 7) described in Japanese Patent Publication No. 57-5520 K: et al.
Peroxidase of microbial origin (EC1, 11, 1, 7) described in Japanese Patent Publication No. 58-5035, etc. can be used. Among these, non-specific peroxidases of plant or microbial origin are preferred. An example of a preferred peroxidase is horseradish peroxidase (
Optimum pH about 7,0), radish peroxidase,
Cochliobolus mlyabeanus peroxidase (optimum pH 5, 0-5, 3), Pe1
licularia filamentosa peroxidase (maximum pH 4.7 to 4.9) and the like.

The oxidase may be any oxidase that oxidizes the test substance with oxygen (02) to generate H2O2. Oxidase can be selected depending on the test substance.

Specific examples of oxidases that can be used in the present invention include the following.

Glucose oxidase (ECI, l, 3.4; optimum pH approximately 5.6) Cholesterol oxidase (EC1, 1, 3, 6, optimum pH approximately 5.8) Uricase (ECI, 1, 3, 3; optimum pH approximately 5.6) Approximately 7.5-8.0
) Sarcosine oxidase (EC1,5,3,1, optimum pH about 7.0-8.0) lactate oxidase, pyruhate oxidase, glutamate oxidase, glycerol oxidase, bilirupine oxidase, etc.

In addition to this, there is also JP-A-53-24893 Gin, JP-A-56-4.
5599, Japanese Patent Application Laid-open No. 157-208998, Japanese Patent Application No. 57-165233, etc., oxidases or combinations of multiple types of enzymes containing oxidases can be used. Cofactors and/or coenzymes can be used in combination with oxidase as needed.

As the wood-philic polymer binder used in the reagent layer, Japanese Patent Publication No. 53-21677 Gin, Japanese Patent Publication No. 45599-1988, Japanese Patent Publication No. 5519-1987, and Japanese Patent Publication No. 164356-1983 are used.
The binder can be appropriately selected from known wood-philic polymers used as wood-philic polymer binders in the reagent layer of multilayer analytical elements described in Japanese Patent Application Laid-Open No. 57-208997. An example of a hydrophilic polymer is gelatin (
e.g., acid-treated gelatin, deionized gelatin T), gelatin, derivatives (e.g., phthalated gelatin).

hydroxymethyl acrylate grafted gelatin, etc.)
, pullulan, pullulan derivatives, agarose, boribicol alcohol, polyvinylpyrrolidone, polyacrylamide, and the like. Among these, gelatin is common and preferred.

The dry thickness of the reagent layer ranges from about 5 #Lm to about 60JLm, preferably from about 10JLm to about 30 pLm. The content of peroxidase in the reagent layer, rij, is approximately 5
The range is from 1,000 U/mI to about 10,000 U/m'', preferably from about 1 U/m' to about 60,000 U/rri'. The content of oxidase in the reagent layer varies depending on the type of oxidase, It generally ranges from about 1,000/m' to about 100,000 U/m', preferably from about 3,000 U/rn' to about 50,000 tJ/rn'.In the case of glucose oxidase, the content is about 2,000/m” to approximately 40,000 U/m”
me, preferably about 4,000 U/m” to about 30,000 TJ
/m' range. The content of the hydrogen peroxide indicator in the indicator layer can be determined as appropriate depending on the expected content of the test component contained in the aqueous liquid sample.

As an embodiment of the present invention, the reagent layer contains a hydrogen peroxide indicator and peroxidase, and the oxidase layer contains oxidase on the ":" side of the reagent layer (the side opposite to the support with respect to the reagent layer, that is, the side far from the support). and oxidase were added to the porous IJ (interlayer,
Contained in either the constant area porous layer, adhesive layer, or light shielding layer 1
There is a certain way to make it happen. JP-A-57-2089971: As described and arranged in the publication, oxidase is placed above a reagent layer containing a hydrogen peroxide indicator and peroxidase (hereinafter, when this layer is specifically designated, it is referred to as an indicator layer). The mode of inclusion in the layer allows the oxidation reaction catalyzed by oxidase to proceed smoothly and quickly due to the efficient diffusion and transport of the airborne fluorine necessary for the oxidase-catalyzed substrate reaction to the oxidase. This is one of the preferred embodiments of the present invention because highly effective illumination of color development or shortening of analysis time can be achieved. When oxidase is contained in a layer on the side of the reagent layer, the content value of oxidase may be the same as the above-mentioned content, and the oxidase layer can be provided directly on the side of the indicator layer or via an intermediate layer described below. can.

The reagent layer contains a pH value close to the optimum pH of oxidase and peroxidase, or at least within a range in which the activities of both enzymes are not substantially inhibited and the color development (or color change) reaction of the hydrogen peroxide indicator proceeds smoothly and quickly. pH value,
That is, under the analysis conditions, the pH value of the reagent layer is approximately 4.0.
to 7.5, preferably from about 4.5 to about 7.0. When the oxidase layer is provided above the reagent layer, or when the oxidase is contained in any layer above the reagent layer, the oxidase layer or the layer containing oxidase is provided with a pH value at or near the optimum pH value for the oxidase. The indicator layer contains a buffer that maintains the peroxidase at or near the optimum pH value, or at least the activity of the peroxidase is not substantially inhibited.
In addition, a pHH buffer can be included to maintain the pH value at which the color development (or color change) reaction of the hydrogen peroxide indicator proceeds smoothly and quickly. As a pH buffer, rBioch
emistryJ, 5(2), 4B? -477 (+9
1(6), R.

Edited by M. C. Dawson et al.
or Biochemical Research J 2nd
Edition (Oxford at the C1arendon
PreSS, published in 1968) 47G-508j:f
,,rAnalytical Biochemistr
yJ, 104.300-310 (1980), “Basic Chemical Handbook” edited by Kuchiki Kagakukai (Tokyo, Maruzen, 1866)
Published in 2013) pp. 1312-1320, edited by Japanese Flower Association [
It can be appropriately selected and used from the known pH buffers described in Biochemical Data Book IJ (published by Tokyo Kagaku Dojin Co., Ltd., 1979), pages 17-24, Japanese Patent Publication No. 57-28277, and the like.

An oxygen permeable and protein impermeable light shielding layer (hereinafter sometimes referred to as a light shielding layer) is submerged in -1- of the reagent layer (or oxidase layer). Oxygen-permeable and protein-impermeable means that this layer is not permeable under analytical conditions, i.e. when water, the solvent of an aqueous liquid sample or blood sample, penetrates this layer and causes the layer to mix or swell. Oxygen in the air (o
2) means that it is essentially transmissible, whereas the normal white matter is virtually transmissible. Also, protein here refers to protein in the sense of moderate cooling, which has molecules -r,) of about 5,000 or more, and especially hemoglobin (about 65,000 molecules).
It is a complex protein that has the activity of degrading hypermorphic hydrogen, such as heme proteins represented by , catalase (molecules, approximately 250,000 molecules), etc. The oxygen-permeable, protein-impermeable light-shielding layer is made up of fine titanium dioxide powder that has light-shielding and light-reflecting properties dispersed in a hydrophilic (or weakly hydrophilic) polymer binder that has the ability to form a film of oligoacid. It is a substantially non-porous layer. The light-shielding layer blocks the color of a blood sample spotted on the developing layer, which will be described later, when measuring color development or discoloration in the reagent layer by reflection photometry from the light-transmitting support side, especially the red color caused by hemoglobin in the case of whole blood. At the same time, it functions as a light-reflecting layer and a background layer.

The fine titanium dioxide powder used in the light shielding layer is aluminum oxide (alumina, A4120g) or hydrated oxide of aluminum (e.g., alumina hydrate A203IIH2).
0, A standing 203.3) 120 etc.) Aluminum compounds containing trivalent aluminum and shochu, shi, or valent aluminum and other bases, V.

Titanium oxide fine powder that has not been surface treated (mainly VA) with a substance containing (e.g., tetravalent F1 element) and acid sulfur, or titanium dioxide fine powder that has not been surface treated (
(Unspecified), these are collectively referred to as titanium dioxide fine powder without alumina treatment. Titanium dioxide fine powder is anatase type, rutile (
Rutile type, or Brookit type.
Type e) Any crystal type may be used.

The average particle size of the fine powder ranges from approximately 0.1 μm to approximately 1.0 μm as a commercially available product.
i ranges from about 0.157 zm to about 0.5 μm. JL examples of fine titanium dioxide powder without alumina treatment include 1'[titanium oxide powder without surface treatment, 11-dioxide titanium powder surface-treated with titanium hydroxide, and silica powder.
There are fine titanium dioxide powders surface-treated with silicon dioxide), and among these, fine titanium dioxide powders that are not surface-treated are preferred.

Examples of the wood-philic (or weakly hydrophilic) polymer binder having film-forming ability include gelatin (e.g., acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (e.g., phthalated gelatin, hydroxymethyl acrylate-grafted gelatin, etc.), polyvinyl alcohol, regenerated cellulose,
Cellulose acetate (e.g. cellulose diacetate)
Among these, gelatin, gelatin derivatives, etc. are preferred. Gelatin and gelatin derivatives can be used together with known hardening agents (crosslinking agents). In addition, when these polymers are used in the adhesive layer described later, the polymer binder of the light shielding layer can be selected from a wide range of wood-loving polymers as in the case of the indicator layer.

The ratio of fine titanium dioxide powder without alumina treatment to the polymer binder (when dry) in the light shielding layer is such that the light shielding layer is non-porous to the extent that oxygen permeability is maintained and at the same time protein impermeability is maintained. (This is smaller than the average pore size in which a developing action or a metering action appears in the porous developing layer, and also includes micropores +1 that do not have an expanding action or a metering action.) can. Specifically, this is a ratio of about 0.6 to about 18 degrees, preferably about 0.8 to about 1. The range is 5.

The dry explosion thickness of the light shielding layer is about 3 pm to about 30 pm.

Preferably it ranges from about 5 μm to about 20 μm.

An intermediate layer can be provided between the reagent layer and the light shielding layer, or between the reagent layer and the oxidase layer if an oxidase layer is provided, or between the oxidase layer and the light shielding layer, as necessary. . As the intermediate layer, a layer of a wood-philic polymer having the same film-forming ability as that used for the reagent layer can be used. The thickness of the intermediate layer is approximately 0.2#1. m to about 1 opLm, preferably about 0.5 μm to about 7 μm. An adhesive layer can be provided between the light shielding layer and the porous spreading layer described below, if necessary.

The adhesive layer is a hydrophilic layer that has the same film-forming ability as that used for the reagent layer and can adhere and form a porous spreading layer when wet with water or swollen with water. P
1 polymer can be used. The thickness of the contact layer ranges from about 0.5 μm to about 20 am, Df, preferably from about 1 μm to about 10 μm. Typical and desirable hydrophilic polymers used in interlayers and adhesive layers include gelatin, gelatin derivatives, polyacrylamide, and polyvinyl alcohol.

When a reagent layer, a light shielding layer, an intermediate layer, an adhesive layer, and an oxidase layer are provided, the oxidase layer and indicator layer may contain a surfactant as necessary.

The surfactant is preferably a nonionic surfactant, particularly a nonionic surfactant having a chain structure of 8 to 15 oxyethylene or oxypropylene.

In addition to this, a curing agent (crosslinking agent) is added to these layers as necessary.
, a softener, a plasticizer, and other known additives.

A porous spreading layer or a constant area porous layer (patch) is provided on the light shielding layer, either directly or via an adhesive layer. For porous 114 open layer (hereinafter also referred to as "open layer"), Japanese Patent Publication No. 53-21677 is used.

JP-A-55-c+og5cz-n-1 JP-A-58-123
458, etc., Kasakata porous media layer, woven fabric 11G open layer described in JP-A-55-164356, JP-A-57-66359, etc., JP-A-57-148250 A layer consisting of paper containing the polyolefin polymer filament pulp described, etc. can be used. As a constant area porous layer, it is described in Utility Model Application Publication No. 57-42951 etc. 11
Among these, 1) (interlayer) is preferable, and as a l5fc open layer, a single layer of membrane filter (planche polymer layer), polymer beads are glued with a polymer adhesive that does not wet with water. The three-dimensional lattice granular structure layer formed by contact bonding is preferably a woven IJG open interlayer. can.

The porous spreading layer may contain a surfactant, preferably the above-mentioned nonionic surfactant, if necessary. Furthermore, for porous 11G open layer,
As described in No. 1, a part of a reagent containing an enzyme such as cholesterol esterase and a pH buffer can be included. Further, fine titanium dioxide powder without alumina treatment can be dispersed and contained in the porous 1+4 open layer.

The multilayer analytical element of the present invention includes at least one reagent layer or one layer on the "-" side of the reagent layer (i.e., the side far from the support when viewed from the reagent layer) (a porous partition layer or a porous layer with a constant area porosity). A compound containing a cation that can form a poorly soluble Fq4 in water with a fluorine anion (including a layer) (hereinafter referred to as a poorly soluble F salt-forming compound) can be contained. Here, "poorly soluble in water" means that the solubility per 100 g of wood at 25° C. is 0.2 g or less. As the poorly soluble F salt forming compound, the poorly soluble F salt forming compound described in Japanese Patent Application No. 57-131750 can be used. The cation contained in the poorly soluble F salt-forming compound is Ca.
' and Mg2+ are preferred since they also have a stabilizing effect on enzymes such as oxidase and peroxidase. The counter anion is a lower aliphatic monocarboxylic anion.

Lower aliphatic dicarboxylic acid anions, hydroxycarboxylic acid anions, halogen anions, phosphorus-resistant anions, etc.
It's so sad. Hardly soluble MF11! As one compound for the formation, acetate chloride J. is preferred. The hardly soluble +l F salt-forming compound is a porous spreading layer, a constant area porous Pi layer, an adhesive layer, a light shielding layer, a reagent layer, and when an oxidase layer is provided, the oxidase layer, an intermediate layer, etc. Or...
It is preferable to include it in one or more of the adhesive layer, light shielding layer, and oxidase layer. Containing poorly soluble F II forming compound 11
1 is 0.1meq to le per multilayer analysis element 1rrf
q, preferably in the range of 0.2 meq to 0.5 eq.

Japanese Patent Application No. 57-131750-; According to the method described in Specification 7, a poorly soluble F salt-forming compound is dissolved or dispersed in a coating solution for a specific layer and applied, or a porous spreading layer is applied to the adhesive layer. By dissolving a sparingly soluble F j1-forming compound in the dampening wood supplied to the adhesive layer during lamination, and supplying the dampening water to the adhesive layer, the sparingly soluble F salt-forming compound is added to the multilayered '7 analysis material. It can be used in summer.

In the multilayer chemical analysis material of the present invention containing a poorly soluble +l:'F salt-forming P1 compound, the fluoride content of the blood sample is approximately t5m from O in terms of NaF.
g/ml blood sample range), virtually the same analyte measurement efficiency as with fluoride-free surface fluid samples.
The more that i is obtained, the more the interference (or interference) effect by fluoride is divided by D1, so the embodiment containing the poorly soluble F salt-forming compound is an extremely preferred embodiment of the present invention.

The integrated multilayer chemical analysis element prepared by laminating and integrating the above-mentioned layers was cut into an appropriate size, and
No. 6079, Utility Model Publication No. 142454, 1983, Japanese Patent Application Publication No. 1983
It is convenient to use it as an analysis slide by storing it in the slide frame described in Japanese Patent Publication No. 58-50114'4, Japanese Utility Model Application Publication No. 58-32350, etc. In addition, the multilayer analysis element can also be used in the form of a long tape or by being attached to an aperture card or the like.

Although the explanation so far has been about an integrated multilayer chemical analysis element in which all layers are bonded, the present invention is not limited to this, and the present invention is not limited to this, but can be applied to an integrated multilayer chemical analysis element in which any of the layers are bonded. It can also be applied to

Quality revision of mI description using the multilayer analysis element of the present invention.

and rcIinical ChemistryJ,
24 (8), +335-1342 (+!37!])
Mainly based on colorimetric analysis/, l
Quantitative analysis of a test substance in an aqueous liquid sample can be carried out using the following principle.

Since the multilayer chemical analysis element of the present invention contains an enzyme,
When carrying out the analysis, the temperature range is 25°C to 45°C, preferably 30°C to 40'C, for 3 minutes to 30°C.
For those who perform colorimetric analysis after incubation for 0 minutes, preferably 4 to 15 minutes.

(end point method) or 5 years old or 5 years old or 2 times or more at a fixed time interval after the induction period of the reaction at the same temperature.I1J: (rate method) It can be implemented.

The present invention will be specifically explained by the following examples.

[Reference Example 1] (A) Titanium dioxide fine powder surface-treated with A-203,
After dispersing 5 g of each of (B) fine titanium dioxide powder surface-treated with the mixed oxide of A 203/5i02 and (C) fine titanium dioxide powder without surface treatment in 40 ml of water, 1 g of NaF was added to each. In addition, the dispersion was further stirred in a stirring forest coated with Yg polytetrafluoroethylene, and after several hours had passed, the p and H values of the dispersion were measured, and the results shown in Table 1 were obtained. Note that the same operation was performed as a blank without adding fine titanium dioxide powder.

1 no, (::r< White Table 1 T i O2 fine anatase rutile without addition powder crystal form (0.15 ~ (020 ~ (bura (particle size) 0.
25km) 0.35cm) Surface treatment AC
AB - pH value 11.5 8.4 11.7 11.3 8.
0 From the results in Table 1, A standing 203, A writing 203・S i
The pH value of the titanium dioxide fine powder aqueous dispersion that has been surface-treated with either O2 becomes significantly higher, but the pH value of the titanium dioxide fine powder aqueous dispersion without surface treatment remains at a pH value that is not much different from the blank. That is clear.

[Reference Example 2] AJlzOa (alumina) surface-treated titanium dioxide fine particles (anatase type, size 0.15 to 0.25 μm)
) and titanium dioxide fine particles without surface treatment (anatase type 1 particle size 0.15-0.25 gm) 10 each
A dispersion prepared by mixing and dispersing 1 ton of gelatin, 1 ton of gelatin, and 20 parts by weight of polyethylene terephthalate with a thickness of 180 ILm and undercoated with gelatin) (
Two types of titanium dioxide/gelatin coatings were prepared by coating the film to a dry layer thickness of 5 JLm and drying. 1 OJL of NaF-containing aqueous solution and NaF-free wood (blank) shown in Table 2 were spotted on each coating film, absorbed into the coating film, and left at 25°C for 5 minutes in a sealed box. death.

Electrode for surface pH measurement immediately (manufactured by Toa Denpa Kogyo ■: G5-
165F) was applied to the coating film and the pH value of the wet surface of the coating film was measured, and the results shown in Table 2 were obtained.

Table 2 below is the margin pH of the surface of wet application 11 Aqueous solution (7) NaF O5101520 containing 1ij (blank) (mg/l water) Alumina surface treatment TiO27, 48, 08, 79, 29, 8 No surface treatment TiO27 , 47, 47, 47, 57, 8 In titanium dioxide fine powder/gelatin coated film without surface treatment, Na
The pH value of the coated film as the NaF content of the F aqueous solution increases is small, especially when the NaF content is competitively added to blood samples.
It has the characteristic that no change in pH value is observed within the range of 0 mg/ml.

On the other hand, alumina surface treatment titanium dibutyride/gelatin coating/I
In tl, NaF-containing ti1. As the pH of the coating film increases, the pH of the coating film (1) I: 'j/ becomes stronger, and NaF
Contains a significant amount of "niga" with a pH value within the range of ~10 mg/m.

[Example 1] A reagent layer for glucose measurement having the following composition was placed on a colorless transparent polyethylene terephthalate (PET) (i) scrap film with a thickness of 180 gm and coated with gelatin so that the dry layer thickness was approximately 151 Lm. It was coated using an aqueous solution and dried.

Peroxidase 25000IU Glucose oxidase 100OOIU 1.7-hydroxynaphthalene 5g 4-aminoantipyrine 5
g Gelatin 200g Polyoxyethylene nonyl phenyl ether 2g On top of the reagent layer for glucose measurement, apply an oxygen permeable protein impermeable light shielding layer having the following composition to a dry thickness of approximately 7 pm using an aqueous dispersion. It was dried and set aside.

100g of bi-decorated titanium fine powder without surface treatment (anatase type 1 particle size 0.15-0.25gm) Gelatin log On top of the light shielding layer, apply an adhesive layer of the following tA composition to a dry layer thickness of approximately 2μ
An aqueous solution was coated so as to have a thickness of m and was dried.

Gelatin 4 g Polyoxyethylene nonylphenyl ether O, 1 g Next, water was supplied to the entire surface of the adhesive layer at a rate of about 3 og/m' to moisten it, and then a 100% cotton broad fabric (10
An integrated multilayer chemical analysis element for glucose determination was prepared by laminating the sample (0 double broad) under extreme pressure and drying it.

[Comparative Example 1] Al2O3 was used as the titanium dileprone fine powder used in the light shielding layer.
・An integrated multilayer chemical analysis element for glucose constant was prepared in the same manner as in Example 1 except that S+02 surface-treated titanium dioxide fine powder (rutile type 1 particle size 0.20-0.35 gm) was used. .

Human blood g11 with different NaF contents shown in Table 3 was added to each developed layer of the integrated multilayer chemical analysis element for glucose fixation of the present invention and for comparison obtained in this manner.
Apply 0p1 and leave it in a sealed container at 37℃ for 10 minutes (
Inkvation) and immediately 15! The PH value of the moistened surface of the spreading layer was measured using a G5-165F surface PH electrode, and the results shown in Table 3 were obtained.

Below is the margin Table 3 pH value of the surface of the wet spreading layer Human plasma 0 5 +0 15 2ONaF included; 1
1 (blank) (mg/ml water) Multilayer analytical element of Example 1 5.765.90 5. ! 30 5.9
5 6.05 (Invention) Multilayer analysis element of Comparative Example 1 5. ? fl 8.75 7. +5 7.
42 7.73 In the multilayer analytical element for glucose determination ♀ of the present invention having a light shielding layer containing fine titanium diMide powder without surface treatment, the pH value of the developed layer increases as the NaF-containing stars in plasma increase. J: 0-10 m, which is a NaF-bearing star with low ′jl, especially in the range added to blood samples.
The pH of the developing layer in the range of g/m plasma (
1-' of ft; I is small. On the other hand, A intersection 203・
In the multilayer analytical element for glucose foot movement of Comparative Example 1, which has 41 light shielding layers containing 5i02 surface-treated titanium dioxide fine powder, the pH value of the Is<interlayer increases markedly as the NaF content of plasma increases. Containing star O~10m
Within the range of g/m plasma! ＋＜pH value of interlayer」
The nigga is noticeably larger.

[Example 2] An integrated multilayer analytical yellow for a glucose constant was prepared in the same manner as in Example 1 except that the following composition was adopted as the composition of the adhesive layer.

Gelatin 4g Lucium resistant 1.8g Polyoxyethylene nonylphenyl ether 0.1g [Comparative Example 2] A1203 as titanium dioxide fine powder used in the light shielding layer
・5i02 surface treatment Titanium dioxide fine powder (rutile type, particle size 0.20-0.35 μm) was used, and the composition of the adhesive layer was the same as that of Example 2. Glucose determination for comparison in the same manner; l! An integrated multilayer chemical analysis element was prepared.

The two types of elements thus prepared were each 15 mm thick.
Cut into square chips of x 15mm, JP-A-57-
A chemical analysis slide for glucose determination RT (named the chemical analysis slide of the present invention and the chemical analysis slide for comparison, respectively) was prepared by placing the slide in a plastinum mount disclosed in No. 63452.

On the developed layer of each of the two types of chemical analysis slide IS, 10 JL of human plasma with different stars as shown in Table 14 was spotted, and left in a sealed container at 37°C for 6 minutes (incubation). Immediately, the pH value of the wetted surface of the spreading layer was measured using a G5-165F surface pH electrode, and the results shown in Table 4 were obtained.

In addition, using a chemical analyzer capable of incubation at 37°C for 6 minutes on two types of chemical analysis slides on which the same plasma LOI, LOI was spotted, reflection photometry was performed from the PET film side using It optical light with a center wavelength of 500 nm. Glucose-containing Ja in plasma was measured by a colorimetric method, and the results shown in Table 4 were obtained.

Table 4 Human blood +1 0 5 10 158aF-containing lit, (blank) (mg/ml plasma) Chemistry of the present invention) Slide Jl (interlayer pH value 6.40 8.42 fl, 39
fl, 41 glucose l=degree measurement value (mg/dl) +01 101 100! pH value of chemical analysis slide development layer for comparison B, 40 8.49 7.32 7.
71 Glucose concentration measurement (IIg/dl) 102 10! lit
All 112 In the case of the chemical analysis slide for glucose foot H,1 of the present invention having a light shielding layer containing titanium dioxide fine powder without surface treatment, the i
Substantially the same glucose content f:j
(A11l list price) is 11t. On the other hand, A sentence 203φ
In the case of a chemical analysis slide for comparative glucose measurement using a light shielding layer containing SiO2 surface-treated titanium dioxide fine powder, the
Interference (obstruction) appears, especially in the range where NaF-containing 1 ink i exceeds 5 mg/m mixed plasma.

[Example 3] An indicator layer having the following composition was applied to a colorless transparent polyethylene terephthalate (PET) smooth film with a thickness of 180 μm and coated with gelatin to a dry layer thickness of about 15 pL.
It was coated using an aqueous solution so as to have a thickness of m and dried.

Peroxidase 25,000 IU 1.7-hydroxynaphthalene 5 g 4-aminoantipyrine 5 g Gelatin 200 g Polyoxyethylene nonylphenyl ether 2 g A glucose oxidase layer with the following composition was added to the fingertip layer so that the dry layer thickness was about 2 m. It was coated using an aqueous solution and dried.

Ceratin 4.6 g Glucose oxidase 4000 IU 3.3-Dimethylglutaric acid 0:1 g Polyoxyethylene nonylphenyl ether 0.1 g On top of the glucose oxidase layer, place a ugliness-permeable, protein-impermeable light-shielding layer with the following composition to a dry thickness. An aqueous dispersion was used to coat 41 coats to a weight of about 7 gm and dried.

100 g of fine titanium dioxide powder without surface treatment (anatase type, particle size 0.15-0.25 ALm) 10 g of gelatin Add an adhesive layer of the following composition to the light shielding layer to a dry layer thickness of approximately 2 gm.
The aqueous solution was applied and dried.

Gelatin Jog Vinegar Calcium 5g Polyoxyethylene nonyl phenyl ether 0.1g Next, water was supplied to the adhesive layer at a rate of about 30 g/m' to moisten it, and then a +%5i 100% broad fabric ( 100 double broad) was laminated with φV<square, dried and glucose constant: +1-. A Jll-integrated multilayer chemical analysis element was prepared.

Using the integrated multilayer chemical analysis element for glucose constant prepared in this way, the same NaF-containing human plasma and whole blood as in Example 2 were used to analyze glucose
．． As in Example 2, substantially the same glucose content '+i: (All transplanted) was obtained regardless of the NaF contained in plasma or whole blood.

[Example 4] Rutile-type titanium dioxide fine powder (particles 7-020 to 0.35) without surface treatment was used as titanium dioxide fine powder to be added to the light shielding layer.
An integrated multilayer chemical analysis element for glucose determination was prepared in the same manner as in Examples 2 and 3, except that pm) was used, and each was prepared using human plasma with different NaF contents as in Example 2. When we measured the amount,
Substantially the same glucose content (measured value) was obtained irrespective of the amount of N a F contained in the plasma.

Claims (4)

[Claims] (1) A multilayer chemical analysis element in which a reagent layer, a light-shielding layer, and a porous development layer are provided in this order on a water-impermeable, light-transmitting support, in which the light-shielding layer contains aluminum chloride or aluminum. A multilayer chemical analysis element characterized by ¥4 containing fine titanium dioxide powder that has not been surface-treated with a substance containing a hydrous oxide or fine titanium dioxide powder that has not been surface-treated. (2) The multilayer chemical analysis element according to claim 1, wherein the reagent layer or any one layer above the reagent layer contains oxidase. (3) Claim a1, wherein the reagent layer contains a hydrogen peroxide indicator that causes a detectable change in the presence of peroxidase and hydrogen peroxide, and peroxidase.
71111 or 2, multilayering, γ, and analysis summary. (4) The reagent layer or any one layer on the I- side of the reagent layer contains a 4N compound containing a cation that can form a salt that is sparingly soluble in water with a fluorine anion.
The multilayer chemical analysis element according to claim 1.2 or 3.